Rough Timing Guide of Lab Training Session
From 2006.igem.org
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* Making of competent cells | * Making of competent cells | ||
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** Take colony from plate, put in prepared broth, grow over night (O/N) | ** Take colony from plate, put in prepared broth, grow over night (O/N) | ||
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** In morning, take growth, thaw at 0 °C, and purify cells ≈1hr | ** In morning, take growth, thaw at 0 °C, and purify cells ≈1hr | ||
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* Professor Davies safety tour ≈20 min | * Professor Davies safety tour ≈20 min | ||
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* Perform DNA extraction ≈70 min involving: | * Perform DNA extraction ≈70 min involving: | ||
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** Take tray of DNA | ** Take tray of DNA | ||
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** Identify two parts needed | ** Identify two parts needed | ||
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** Re-suspend DNA in buffer with pipette | ** Re-suspend DNA in buffer with pipette | ||
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** Mix with competent cells | ** Mix with competent cells | ||
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** Incubate at 37 °C ≈50 min | ** Incubate at 37 °C ≈50 min | ||
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** Spread on petri dish with antibiotic resistance O/N | ** Spread on petri dish with antibiotic resistance O/N | ||
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* With the cells that grew O/N (prepared by organizational committee), take about 3 mL and place in microcentrifuge tubes | * With the cells that grew O/N (prepared by organizational committee), take about 3 mL and place in microcentrifuge tubes | ||
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* Add glycerol and store in fridge for when we need the cells again | * Add glycerol and store in fridge for when we need the cells again | ||
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* With remaining O/N culture, perform plasmid extraction using specified kit (miniprep) ≈1 hr | * With remaining O/N culture, perform plasmid extraction using specified kit (miniprep) ≈1 hr | ||
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* Prepare two separate tubes with restriction enzyme. One will have needed vector, while the other the purified plasmid. Let enzyme digest ≈1 hr | * Prepare two separate tubes with restriction enzyme. One will have needed vector, while the other the purified plasmid. Let enzyme digest ≈1 hr | ||
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* Prepare gel for electropheresis | * Prepare gel for electropheresis | ||
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* Load both samples (digests) on gel, and run for ≈40-60 min | * Load both samples (digests) on gel, and run for ≈40-60 min | ||
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* Physically cut out (with razor blade) desired BioBrick part and place in new test tube | * Physically cut out (with razor blade) desired BioBrick part and place in new test tube | ||
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* Use gel extraction kit to purify segments ≈1 hr | * Use gel extraction kit to purify segments ≈1 hr | ||
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* You now have sticky ends, therefore place desired parts in same test tube with ligase to do ligation ≈15-40 min (varies on protocol) | * You now have sticky ends, therefore place desired parts in same test tube with ligase to do ligation ≈15-40 min (varies on protocol) | ||
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* With product of ligation, add to competent cells, and perform transformation as before | * With product of ligation, add to competent cells, and perform transformation as before | ||
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Revision as of 20:41, 15 June 2006
This is an outline of preparatory activities that each group is expected to perform while in training. This outline is general, and the timeline will vary with each group’s performance.
Contents |
Before Training Session Begins
The Operational Committee will prepare the following:
- Making of competent cells
- Take colony from plate, put in prepared broth, grow over night (O/N)
- In morning, take growth, thaw at 0 °C, and purify cells ≈1hr
Training Session 1 (≈1.5-2.0 hrs)
The Training Group will complete the following tasks:
- Professor Davies safety tour ≈20 min
- Perform DNA extraction ≈70 min involving:
- Take tray of DNA
- Identify two parts needed
- Re-suspend DNA in buffer with pipette
- Mix with competent cells
- Incubate at 37 °C ≈50 min
- Spread on petri dish with antibiotic resistance O/N
Before Next Training Session Begins
The Operational Committee will prepare the following:
- Take colonies that grew on the petri dish from transformation done by group during last training session, place in broth, and grow O/N
Training Session 2 (≈1.5-2.0 hrs)
The Training Group will complete the following tasks:
- With the cells that grew O/N (prepared by organizational committee), take about 3 mL and place in microcentrifuge tubes
- Add glycerol and store in fridge for when we need the cells again
- With remaining O/N culture, perform plasmid extraction using specified kit (miniprep) ≈1 hr
Training Session 3 (≈4.0-5.0 hrs)
The Training Group will complete the following tasks:
- Prepare two separate tubes with restriction enzyme. One will have needed vector, while the other the purified plasmid. Let enzyme digest ≈1 hr
- Prepare gel for electropheresis
- Load both samples (digests) on gel, and run for ≈40-60 min
- Physically cut out (with razor blade) desired BioBrick part and place in new test tube
- Use gel extraction kit to purify segments ≈1 hr
- You now have sticky ends, therefore place desired parts in same test tube with ligase to do ligation ≈15-40 min (varies on protocol)
- With product of ligation, add to competent cells, and perform transformation as before
--Jovan 22:46, 14 June 2006 (EDT)
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